Human embryonic stem cells (hESC) offer hope that cures can be found for many diseases such as Parkinson's Disease, heart disease or type I diabetes, in which cells of a specific type are damaged or killed. The promise of hESC research is tempered by several significant challenges, not least of which is the difficulty of scaling up culture systems to produce large numbers of undifferentiated and unaltered hESC. Cells at higher passage numbers tend to accumulate karyotypic and epigenetic changes, especially when passaged enzymatically instead of by labor-intensive mechanical means. The abnormalities are linked to the low cloning efficiency of hESC, because the poor survival of single cells or cells in small clumps creates selection pressure in favor of chromosome duplications that confer an adaptive benefit Draperetal.,2004;Andrewsetal.,2005). The stress of hESC dissociation might be eased by reproducing the microenvironment that maintains pluripotent cells within the human blastocyst. This can be done by supplying factors which are specifically expressed in the blastocyst and which maintain the pluripotent inner cell mass. One such factor is FGF2, which is commonly used in hESC medium to suppress differentiation, and is also an autocrine factor released from hESC themselves (Dvorak et al., 2005). Other factors observed to sustain hESC pluripotency are members of the TGF[unreadable] superfamily. Some growth factors, especially TGF[unreadable] superfamily proteins, can be difficult to purify in the active form. Therefore the goal of this project is to over express FGF2 and the TGF[unreadable]-related protein GDF3 in human foreskin fibroblasts, which can be used in co-culture as feeders for hESC. Standard recombinant DNA techniques will be used to develop and test efficient vectors for sustained high expression and secretion in fibroblasts. Once stable lines are established which secrete active factors, hESC will be passaged on the feeders and the undifferentiated and pluripotent state of the cells will be rigorously assessed. In addition, the feeders will be appraised for the ability to support clonal growth of isolated hESC. Thus the ultimate result of this project should be feeder cells which uniquely support the expansion of hESC with a reduced frequency of genotype alterations. [unreadable] [unreadable] [unreadable] [unreadable]